1. Field of the Invention
The present invention relates to a scanning multi-photon excitation laser microscope which detects chemical reaction or fluorescence by multi-photon absorption of a sample and a laser pulse width control method for minimizing a pulse width of a laser beam on a sample surface.
2. Description of the Background Art
A multi-photon exciting scanning type laser microscope has heretofore been known as a laser microscope, in which a sample as an observation object is irradiated with an ultra-short pulse laser beam, and which detects chemical reaction or fluorescence by multi-photon absorption of the sample.
A multi-photon excitation phenomenon occurs at a probability which is proportional to n-power (n=2 for 2-photon excitation, n=3 for 3-photon excitation) of a photon density per unit area and unit time. Therefore, a laser beam of an ultra-short pulse usually of a sub picosecond order is used in a light source for a multi-photon excitation method.
However, the laser beam of the sub picosecond pulse does not have a completely single wavelength, and has a wavelength width having correlation with a pulse width. In general, when light passes through an optical system, a speed thereof in a medium is low with a shorter wavelength, and high with a longer wavelength. Therefore, when the pulse laser beam has the wavelength width as described above, a difference is generated in a transmission time in accordance with the wavelength during transmission through the optical system. As a result, so-called chirp occurs in which the pulse width after the transmission through the optical system is expanded as compared with the pulse width before incidence upon the optical system.
The multi-photon excitation phenomenon depends on a photon density. Therefore, expansion of the pulse width on a sample surface caused by the chirp deteriorates the probability at which the multi-photon excitation phenomenon occurs. As a result, obtained fluorescence is darkened.
It is known that pulse width adjuster, that is,. so-called pre-chirp compensation is used as means for solving the problem. The pre-chirp compensation is an optical system configured by a prism pair, a grating pair, or a combination of these pairs. In the pre-chirp compensation, when a long wavelength light of pulse laser is incident behind a short wavelength light, the expansion of the pulse width after the transmission through the optical system is corrected.
On the other hand, a case in which the multi-photon excitation method is used in the scanning laser microscope. The scanning laser microscope usually includes one selected from a plurality of objective lenses and other optical systems (such as prism and mirror). These plurality of objective lenses and other optical systems differ from one another in optical path length and material. Therefore, the expansion of the pulse width differs with the selected optical system such as the objective lens. In order to perform the multi-photon excitation method on an optimum condition, it is necessary to adjust the pre-chirp compensation, every time the optical system such as the objective lens is selected.
The expansion of the pulse width also depends on the pulse width of the pulse laser beam. Therefore, when the wavelength of the pulse laser beam is variable, the pre-chirp compensation needs to be similarly adjusted for every change of the wavelength.
Methods disclosed in Jpn. Pat. Appln. KOKAI Publication No. 10-318924 and the Related U.S. patent application Ser. No. 09/265183 have heretofore been known as a method for optimizing the adjustment of the pre-chirp compensation.
Among these, the Jpn. Pat. Appln. KOKAI Publication No. 10-318924 discloses a method of: disposing an apparatus comprising a collimating optical system disposed opposite to the objective lens via the sample and an autocorrelator for measuring the pulse width; and adjusting the pre-chirp compensation while the pulse width on the sample is measured.
Additionally, in the method, since the collimating optical system and autocorrelator are disposed in the vicinity of the sample, the apparatus is enlarged in size. Therefore, for example, patch clamping is performed by inserting an electrode into the sample during observation. In this case, a problem occurs that it is difficult to secure an operation space around the sample.
Moreover, the Related U.S. patent application Ser. No. 09/265183 discloses a method of disposing a correction optical member in accordance with the optical path length of each objective lens, and selecting and disposing the correction optical member in the optical path in accordance with the selected objective lens.
In the method, when there are other selected optical systems such as the prism and mirror in addition to the objective lenses, the correction optical members are necessary for the optical systems, and the apparatus therefore increases in size. Moreover, it is also difficult to prepare a large number of correction optical members for all the optical systems. Furthermore, the correction optical members are only changed, and fine adjustment cannot be performed. Therefore, when the laser beam with a variable wavelength is used, it is also difficult to optimize/adjust the pre-chirp compensation in all wavelength areas.